Cryofixation is a sample preparation method frequently used in electron microscopy. In it, a water-containing sample is frozen very quickly (cryofixed) to a temperature of less than −150° C., i.e. it is cooled very rapidly in order to avoid the formation of ice crystals. Cryofixation has proven to be particularly suitable for investigations of structural biology. The objects to be investigated, for example cells, enzymes, viruses, or lipid layers, thereby become embedded in a thin, vitrified ice layer. The great advantage of cryofixation is that the biological structures can be obtained in their natural state. For example, a biological process can be halted at any point in time by cryofixation, and investigated in that vitrified state, for example in a cryo-electron microscope but also in a light microscope with corresponding sample cooling; cryo-light microscopy serves principally to locate relevant regions in the sample, which can be noted and then viewed in more detail in a cryo-electron microscope.
The frozen samples, which as a general rule are located on electron-microscope sample carriers known per se, for example a grid or a pin stub mount for scanning electron microscopy, must be conveyed (under the aforesaid cryogenic conditions and with water excluded) into corresponding sample carrier mounts that can subsequently be conveyed, in appropriate holders, into the aforesaid microscopes. A typical sample carrier mount for use in connection with the present invention has been disclosed, for example, by the document U.S. Pat. No. 8,395,130 B2, in which a grid that constitutes a sample carrier and carries the frozen sample can be immobilized with clip elements in a corresponding mount.
What have hitherto been used for this are fairly improvised solutions in which liquid nitrogen is stored, for example, in a Styrofoam container in which the requisite manipulation steps for conveying the grid into the sample carrier mounts were performed. The formation of cryogenic nitrogen gas from the liquid nitrogen on the one hand ensures the necessary low temperature and on the other hand creates an anhydrous atmosphere in the Styrofoam container, so that contamination of the samples with water, and consequently with ice crystals, could be prevented.
In order not to impair the quality of the frozen samples, it is very important that they be transferred in cooled and contamination-free fashion between the processing units being used, for example the cryofixation device, freeze fracture apparatus, and coating apparatus, and the analytical devices, in this case principally a cryo-light microscope and cryo-electron microscope. For this as well, in everyday laboratory practice it has hitherto been usual to resort to fairly improvised solutions or to specifically fabricate loading and transfer systems in-house.
A vacuum cryotransfer system is represented, for example, by the “Leica EM VCT100” system of the manufacturer styled Leica Microsystems, with which a sample carrier mount can be removed from a manipulation container cooled with liquid nitrogen and conveyed into the observation chamber, likewise cooled with liquid nitrogen, of an electron microscope. This system is relatively complex, however, since it is a vacuum system that offers only a limited manipulation clearance and entails relatively complex handling of the corresponding samples in the manipulation container. What is necessary for cryo-light microscopy, however, is relatively simple handling of the manipulation container, in which all that is desirable is simple cooling and replacement of water with liquid nitrogen that is introduced into the manipulation container, principally in order enable flexible incorporation of a manipulation container of this kind into various analytical systems and, in particular, microscopy systems.